We are utilizing molecular and genetic approaches to examine the generation of diversity of the muscle protein myosin heavy chain (MHC) and to define the functional significance of this diversity. We have previously shown that Drosophila melanogaster has a single gene coding for muscle MHC. Alternative splicing of the RNA coding regions (exons) from this gene may lead to the production of up to 480 alternative forms (isoforms) of the MHC protein. During the forthcoming period we propose to examine the diversity of MHC isoforms by isolating and analyzing cDNA clones made from larval and adult RNA and by observing the hybridization pattern of alternative exon- specific oligonucleotides to tissue sections from organisms at these stages of development. We will also determine the molecular defects in several flightless mutants which fail to synthesize MHC in their thoracic muscles. This will serve to define regions of the gene that are important for muscle-specific protein accumulation. We will utilize cDNA clones along with the MHC gene promoter to develop vectors that express high levels of individual isoforms. These vectors will be inserted into the germline of mutants that are unable to synthesize MHC in the bulk of their thoracic muscles. MHC isolated from the thoraces of these transgenic flies will be biochemically and functionally analyzed in order to determine the distinct properties encoded by alternative exons. In another series of experiments we will examine the in vivo function of specific alternative regions of the MHC protein by constructing MHC genes that lack a specific alternative exon and inserting these genes into mutants which fail to synthesize either thoracic MHC or all MHC isoforms. We will examine muscle function and ultrastructure in these transgenic organisms. The presence of a single MHC gene in Drosophila, the availability of MHC mutants, and the use of vectors capable of expressing the MHC gene via germline transformation makes this a powerful approach to examining the functional diversity of the MHC protein and should lead to insights into how the contractile apparatus functions in normal and mutant muscles. Funds are sought for two undergraduates and one graduate student. Undergraduates will have a two year training period in which they will first be closely supervised by a postdoctoral fellow or advanced Ph.D. student. This will be followed by an independent research project designed in conjunction with the P.I. The graduate student will receive training directly from the P.I. and other laboratory personnel and learn to be an independent scientist. All students will be required to attend weekly laboratory meetings, journal clubs and seminar.